1a. Optimizing immune reconstitution after HLA matched sibling SCT T cell depletion: In 2012 we initiated a new T cell depletion study to address some of the shortcomings of an earlier trial 07-H-0248. In this protocol 12-H-0028 the T cell depletion technique was changed so as to negatively select for CD34 cells using a CD3 CD19 antibody magnetic separation approach from Miltenyi Corporation under an investigator IND. The transplant product contains a mixed population of CD34 cells more mature progenitors and NK cells. It was anticipated that hematological reconstitution would be more rapid and robust with this method. Subjects received myeloablative conditioning with cyclophosphamide (60 mg/kg/dose x 2), fludarabine (25 mg/m2/dose x 5) and total body irradiation (12 Gy divided in 8 fractions, with lung shielding to 6 Gy). (ii) No planned donor lymphocyte infusion DLI given; ( DLI was reserved only for patients with graft failure or relapsed leukemia) (iii) Older patients receive 600cGy instead of 400cGy to improve engraftment since a number of older patients had engraftment failure in the previous protocol. This protocol accrued 20 patients and is now closed. Hematological recovery was prompt. However we noted increased frequency of grade II-IV acute graft-versus-host disease (GVHD) and graft failure requiring second transplantation in four. Full analysis of outcome awaits longer follow-up. This trial was terminated because of the high frequency of GVHD and graft failure which precluded its use as a platform for T cell therapy. In 2013 a new T cell depletion protocol 13-H-0144 initiated Peripheral Blood Stem Cell Allotransplantation for Hematological Malignancies Using Ex Vivo CD34 Selection - A Platform for Adoptive Cellular Therapy. This uses a modified version of protocol 07-H-0248 Currently 12 patient have received transplants, 11 survive. Three patients developed acute GVHD grade II-IV. Having satisfied the safety criteria in the first 10 patients this protocol will now serve as the platform for add-back of virus-specific T cells (see below). 1b. Prevention of GVHD in haploidentical donor SCT. In 2012 we completed a study of low dose IL-2 in healthy volunteers to determine the kinetics of Treg and NK expansion. We found a twofold or greater increase in Tregs and also in natural Tregs by 7 days. IL-2 at the lowest dose of 100,000u/m2 daily for 5 days was optimal. This schedule was without significant side effects. On the basis of these results we have obtained IRB and FDA approval to explore the use of LD IL2 in haploidentical SCT. Donors will be haploidentical family members. The transplant will be partially depleted of T lymphocytes to a dose of 1 x 106 CD3 cells/kg. Recipients will receive LD IL-2 from day 2 for 12 weeks. The main study end point will be the development of grade II-IV acute GVHD with stopping rules for unacceptable day 100 mortality. 2a. Prevention of viral infection after SCT. In collaboration with Dr Catherine Bollard (Childrens National Medical Center Washington DC) and Dr David Stroncek (Depart,ment of Transfusion Medicine) we have developed a robust system to expand multivirus specific T cells recognizing CMV, EBV, Adenovirus and BK virus which commonly complicate the outcome after SCT. Using a peptide mix of common immunodominant antigens from these viruses we have induced multivirus specific T cells in high frequencies. The clinical protocol has been approved by the IRB and the clinical grade cell product multi-virus specific T cells (MVST) has been validated and is under review by the FDA for approval under an investigator IND. The objective of this Phase I/II protocol is first to establish safety of the MVST product and second to prevent CMV, EBV, BKV and adenovirus reactivation after SCT. Patients undergoing T cell depleted SCT on protocol 13-H-0144 will be eligible to receive an MVST infusions between day 7-14 post transplant. Viral antigenemia and viral infection will be monitored. To assess a potential economic benefit from MVST, hospital stay will be monitored to determine if MVST reduce post transplant in-patient days. 2b. Role of Th17 T cells in gut mucosal resistance to gram-negative bacteria. Emergence of gram-negative bacteria with resistance to multiple antibiotics is a significant challenge facing the healthcare system. Klebsiella pneumoniae is responsible for approximately 15% of nosocomial infections, predominantly affecting immunocompromised patients and in recent years multi-antibiotic resistant strains have emerged. SCT recipients are especially at risk from these resistant bacteria: In 2011-12 the NIH Clinical Center experienced an outbreak of multidrug resistant K. pneumoniae affecting 19 patients, most of whom had received stem cell transplants, 7 of whom died from the infection. While neutrophils play the predominant role in protection against Gram-negative bacteria emerging evidence suggests that T cells can orchestrate and regulate host defenses, especially at mucosal sites where Th17 cells physiologically reside. Th17 cells and their secreted cytokines bridge innate and adaptive responses and are involved in first line mucosal host. Recently, antigen-specific Th17 T cell recognizing K. pneumoniae have been described in an animal model of mucosal vaccination and infection. We have generated K. pneumoniae specific TH17 T cells and plan to develop a clinical-grade Th17 cells specific for Klebsiella pneumoniae from patients or HLA-matched normal donors for adoptive transfer as a secondary prevention of KPC-Klebsiella bacteremia in immunocompromised patients treated at the NIH Clinical Center. Completion of the proposed study will test a novel strategy of targeting antibiotic-resistant bacterial pathogens that cause nosocomial infections with high mortality. 3. Prevention and treatment of GVHD and post transplant complications with mesenchymal stromal cells: In 2013 we completed a phase I trial using third party, early passage, mesenchymal stromal cells (MSC) generated in the NIH Cell Processing Section for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT where we demonstrated both safety and efficacy of MSC infusions. These results confirmed that MSC are associated with rapid clinical responses of steroid refractory GVHD. We are now initiating a phase II trial evaluating the use of MSC infusions at the onset of systemic steroid treatment for acute GVHD. The objective is to determine the effect of MSC on time to resolution of acute GVHD, progression to chronic GVHD and incidence of steroid refractoriness. All patients undergoing SCT at NIH are eligible for this protocol which will start in 2014. 4. Long term follow-up: Our stem cell transplantation program has now accumulated 20 years of follow-up on protocol 05-H-0130. Currently over 150 patients are being followed long term. Our present focus is on outcomes in the second decade after SCT. We found that males have an increased risk of cardiovascular events and have a cardiovascular age 10 years over their actual age. To further investigate this we studied 16 asymptomatic post allo-SCT survivors (11 males; 5 females) median age of 45 years at transplant who underwent coronary calcium scoring and contrast enhanced coronary CT angiograms at a median follow up of 5 years post transplant. Non-obstructive coronary artery disease was detected in seven (44%) patients. Additionally, four (25%) had aortic root calcification. Coronary artery disease was detected in 4 of 13 (30.8%) low risk patients. We conclude that coronary calcium score with or without CT angiogram is a safe, feasible, highly sensitive study in transplant survivors; and even asymptomatic, low-risk survivors may benefit from screening.